PRE-STAINING IN BOTANICAL MICROTECHNIQUE. THE ALCOHOL-XYLOLSAFRANIN METHOD A FEW years ago the writer had occasion to imbed some small botanical objects in which it was important to section accurately with respect to the median axis. On clearing in xylol, the material became quite transparent, as so frequently happens, making it almost impossible to see the pieces in the paraffin block, much less to orient the material correctly for cutting. In the ribbon it was again difficult to find the small sections or to ascertain if the material was at all suitable for mounting and staining. The difficulties just enumerated are familiar to all technicians, whether small or large objects are dealt with and, as a result, much valuable time is lost, materials are ruined and the finished product is often thrown into the discard. The writer had read somewhere that it was possible to stain materials in bulk to render them more conspicuous, but details were lacking. Probably it was thought to be altogether too simple a procedure to require further elucidation. Inquiries put to several technicians did not elicit much definite information. This is not surprising, since few workers seem to be aware of the many advantages of prestaining in micro-technique. A method of pre-staining was devised which was so generally successful with all kinds of materials and which required so little extra effort that all subsequent imbedding has been done in this way. Not only are imbedding and cutting facilitated, but permanent mounts are also possible without extra labor. The plant parts are killed and fixed in the favorite fluid, and washing, hardening and dehydrating follow in the ordinary way. The clearing is done in alcohol-xylol mixtures, a series of 5, 10, 30, 50, 75 and 100 per cent. xylol in absolute alcohol being generally employed. The stain mixture is inserted in the series in place of the 75 per cent. alcoholxylol. It is prepared as follows: safranin is dis solved in absolute alcohol to make a saturated solution; 100 parts of the alcoholic safranin are mixed with 300 parts pure xylol. Some of the stain will precipitate out, and the mixture may be filtered, although this is not absolutely necessary. The material is run up through the lower percentages of xylol in alcohol through the 50 per cent. mixture and is then put directly into the safranin mixture, where it is left for 24 hours or longer, depending upon the size and quantity of the material to be stained. It will be seen that the material assumes a deep red color, the fluid at the same time becoming somewhat clear. If much material is to be stained, the original solution may be replaced with fresh stain mixture. There is no danger of over-staining. From the stain mixture the material is run through pure xylol. This will bring down additional safranin as a precipitate, which may be removed by an extra washing with xylol. Embedding proceeds in the regular way. This becomes an easy task, even with small objects, and the pieces are easily seen in the paraffin block. Cutting is facilitated, and in the ribbon the sections stand out clearly. The ribbon may be examined under the microscope and surplus and useless sections may be eliminated with certainty at once. Mounting is done with a minimum of albumen fixative, using no more water than is necessary to smooth out the sections. Any excess water is immediately drained off and the slides are thoroughly dried with gentle heat. Twenty-four hours are not too long a time for drying, and an incubator is best used to eliminate dust and to guard against melting the paraffin. The slides may then be finished. paraffin is removed with xylol in the ordinary way. At this stage they may be examined under the microscope. The sections will be found to be beautifully stained, and every detail will stand out against a perfectly clear background. A second elimination of unfit material may be made at this time with great certainty, and the only precaution necessary is to keep the slide wet with xylol during the period of examination. The slides may then be finished up by applying balsam and a cover-glass; or they may be run down through the alcohol series, which removes the stain, and any other staining method pursued. The It will thus be seen that this gives a method by which permanent mounts may be made quickly and easily. It is often desirable to make such preparations for temporary class use, and workers in certain fields will find the method adapted to many uses. Mounts thus made have been kept for months without apparent deterioration, the success being apparently determined by the elimination of all sources of water. If a safranin soluble only in alcohol were possible of attainment, mounts made in this way would probably keep indefinitely. Diaphane has not been tried as a substitute for balsam. The advantages of the method may be summed up as follows: (1) Substitution of the stain mixture for 75 per cent. alcohol-xylol is hardly to be considered as an extra effort. (2) The red objects, no matter how small, are readily visible, even in the hardened paraffin block. (3) Accuracy of cutting is facilitated. (4) The cut sections are quite visible in the ribbon and material may be examined superficially for accuracy of cutting, stages desired, etc. (5) Unfit material is eliminated without further waste of time, and sections of value are not inadvertently thrown away. (6) Critical examination of material may be made in the stage of removing the paraffin. (7) Finished mounts may be made at once, without the necessity of going through tedious processes, and especially the individual staining of slides. (8) Slides so made are fairly permanent. (9) Slides not intended for quick mounting may be destained and subsequently treated to any other staining technique. UNIVERSITY OF WISCONSIN CHAS. H. OTIS A STUDY ON THE LIFE HISTORY OF THE BROAD FISH TAPEWORM IN NORTH AMERICA RECENTLY the committee on scientific research of the American Medical Association made a grant to the writer in support of a field study on the life history of the broad fish tapeworm, Diphyllobothrium latum. This species is a well-known and somewhat serious parasite of man in various regions of the Old World. It was first reported in the United States by Leidy, who studied in 1879 a specimen taken from an immigrant. Other cases which certainly were introduced have been reported from time to time and these records have increased rapidly in frequency within recent years. The first case in the human host unquestionably infected by larvae bred in this country was reported by Nickerson in 1906 from the clinic of Dr. Parker, of Ely, Minn. The patient was a boy only two years old who had never been out of the state and had never eaten imported fish. In 1911 Nickerson published data on 65 cases from Minnesota, including another record of local infection. Other indigenous cases have been reported by Warthin from Michigan, by Becker from Chicago, by Magath and by Riley from Minnesota, and by Lyon from Indiana. In some districts this species has come to be the most abundant and important human cestode, and this abundance is of very recent origin. The European form has been introduced into North America many times as more than one hundred cases in man were recorded up to 1922; the list has grown since then though many cases are still unpublished. In fact in certain regions such instances have become too frequent to justify publication. The ova of the parasites were disseminated by sewage systems and thus fishes in connecting rivers and lakes are infected. The history of the parasite at Lake Geneva (Switzerland) is a striking illustration of the way in which the condition is caused and also corrected. No one has as yet shown that the parasite can find here intermediate hosts and the particular small crustacea functioning as such in Europe are rare or unknown here. Moreover, since no accurate examination has been made of the adult tapeworms taken from man here, it may be that the hosts which were infected on this continent really sheltered a new, similar and yet unrecognized species and not the well-known type found in the Old World. Closely related if not identical species have been reported from other hosts than man in this country, thus by Warthin from the gray fox in northern Michigan, and by Hall and Wigdor from the dog in Detroit. The latter authors regarded the form they described as a new species and named it D. americanum. I have myself seen such a tapeworm taken from a dog at Ely, Minn., by Dr. J. E. Thompson. I have also adult tapeworms of this type collected from bears, in the northwestern United States and in Alaska. The adult specimens from this continent have not been studied sufficiently precisely to justify a positive statement concerning their specific identity with the Old World species. The last larval stage, i.e., the form by which the final host is infected, is known as a plerocercoid and occurs in various fish. These plerocercoid larvae are so simple in structure and so imperfectly known that as yet no one can pass upon their relation to definite adult species. I have often found such larval stages of bothriocephalid tapeworms in fish studied in various regions from the Great Lakes to Alaska. Nickerson also records finding such larvae but states distinetly that in the present state of knowledge it is impossible to determine the species to which they belong. The rapid increase in the number of cases of human infection reported in the United States, the consequent increasing contamination of our streams with probable like increase in infection of fish, and the severe anemia incident to the parasitization of the species in the Old World, no less than the general hygienic and biological interest associated with the problem, make it important to study the situation promptly and in such fashion as to secure exact information on the various aspects of the question. The American parasite in man may be identical with the European species, but, if not, two very similar species are now found side by side in certain regions. At least one of them affects man and either one or both of them also occur in other hosts in these regions. On the abundance and distribution of the parasites in other hosts as well as in man depends the frequency of human cases. The life cycle of the tapeworms in this country must be precisely determined, whether a new species is involved or not, since this life cycle need not necessarily be identical with that reported for Europe. Evidently on the exact history of its varied relations to seasons and hosts depends both the manner and facility with which man is infected, and per contra the methods by which such infection may be regulated. In this connection it is essential to consider not only the last larval host but also the earlier phases of the life cycle as well. This is especially important since the species (Cyclops leuckarti) which in Europe serves as the first larval host is either rare or wanting on this continent. Nickerson first showed that the source of human infection could be traced to a definite lake and Magath later demonstrated the occurrence of infected fish and thus of necessity infected intermediate hosts in a lake in the same general region. It is important to confirm these observations and to extend them to other waters for the purpose of determining the range and frequency of the parasite as well as the number and degree of infection of the intermediate hosts. Field studies are essential in securing the facts in the case and thus in furnishing a safe basis for views as to the probable future history of the parasite and possible means for its control and ultimate eradication. With the purpose of studying the problem on the ground a field party has been organized and will carry on work this summer in northern Minnesota where the parasite has been so frequently reported. This party is directly in charge of the writer. Dr. T. B. Magath, of Rochester, Minn., will collaborate in the investigation and have control of the clinical experiments in particular. Dr. H. E. Essex of the University of Illinois will study the early development of the parasite and carry on the experiments in the field. Helpers will be secured as needed. The U. S. Bureau of Fisheries has undertaken to cooperate by sending an apprentice fish culturist to collect fish and maintain the aquaria. The Mayo Foundation has made a substantial contribution to the enterprise by furnishing reagents, apparatus and other help. The problem will be attacked at once from several different angles. One line of work involves securing eggs of the adult tapeworm, developing them in cultures and employing them in feeding experiments to determine the species of crustacea or other small aquatic organisms which can function as first intermediate hosts. It may also be possible to collect naturally infected crustacea. The second stage in the life history will be sought by feeding such infected crustacea to small fish. While the time may not suffice to allow of full development in such first intermediate hosts, undoubtedly the plerocercoid larvae can be obtained as they have been previously from various fish, large and small. Among such specimens some will probably be sufficiently advanced in development to use in experiments with final hosts. In any event such material when carefully preserved and studied may show characters adequate to differentiate the plerocercoids of one bothriocephaloid tapeworm from those of another species. Here again the conclusions can be tested by feeding experiments with various hosts. The problem can hardly be completely solved in a single summer, even with the varied attack planned. But preserved specimens will afford opportunity for continuing the study in my laboratory during the winter. Efforts will also be made to secure and transport living material so that feeding experiments can be continued there. ZOOLOGICAL LABORATORY, UNIVERSITY OF ILLINOIS HENRY B. WARD SPECIAL ARTICLES THE TOXICOLOGY OF CARBON MONOXIDE THE toxicology of carbon monoxide gas always raises the mooted question as to whether carbon monoxide is poisonous per se or produces all its toxic effects from interference with proper oxygenation of tissues. In all higher animals it has been the general opinion of most pharmacologists that carbon monoxide is poisonous by virtue of its combining with hemoglobin to form CO hemoglobin and thus preventing the hemoglobin from combining with oxygen. The affinity of carbon monoxide for hemoglobin has been found to be some two hundred times greater than its affinity for oxygen. Carbon monoxide gas of itself is commonly regarded as being physiologically inert. Some recent work however seems to indicate that carbon monoxide is not as innocuous per se as it has been supposed to be. Thus Warburg (Biochem. Zeit., 177, pp. 471, 1926) has shown that car of oxygen. Again, Haldane in a recent note (Nature, March 5, 1927, page 352) extended these observations to two higher organisms, wax moths, Galleria mellonella and the cress plant Lepidium sativum. He found that the moths behaved normally in as little as 2 per cent. of oxygen at atmospheric pressure, provided this gas is diluted with nitrogen. When however the oxygen is diluted with carbon monoxide about 16 per cent. of oxygen is needed for normal behavior. With smaller amounts of oxygen carbon monoxide is poisonous. Haldane also found that cress seeds do not germinate in an atmosphere of oxygen containing a certain amount of carbon monoxide. In connection with the above observations the author wishes to call attention to certain observations which he has made and concerning which a brief note was published already (Macht, Blackman and Swigart, Proc. of Exp. Bio. and Med., 1924, Vol. 91, pp. 227). While engaged in the study of the effects of various drugs and toxins on the growth of the seedlings of Lupinus albus studies were made on the growth of such seedlings in weak solutions of blood and hemoglobin. The procedure briefly consisted in growing seedlings of Lupinus albus in upright test tubes containing equal parts of distilled water and a plant physiological solution (Shive) on the one hand, and of other seedlings of Lupinus albus in exactly the same control solution plus small amounts of unknown substances to be studied. The elongation of the straight and well-defined roots was measured accurately in each case. It was found that 1 per cent. solutions of blood give a growth index as compared with a growth in normal nutrient solution without blood of about 72 per cent. Having studied repeatedly the effect of normal blood and normal hemoglobin solutions on the growth of Lupinus albus seedlings in the dark, experiments were made on the growth of the seedlings in solutions of blood containing various amounts of carbon monoxide hemoglobin. Here a new and unexpected observation was made. It was found the solution of carbon monoxide hemoglobin produced a poisonous effect on the seedlings as shown by an inhibition of their growth. The following four protocols will serve as illustrations. EXPERIMENT NOVEMBER 3, 1925 Defibrated blood of a pig was saturated with pure carbon monoxide obtained by the addition of concentrated sulphuric acid to formic acid. A 1 per cent. solution of the normal pig's blood was made in Shive solution as described above; another 1 per cent. solution was made of the blood which was saturated with carbon monoxide. A third solution was made containing 0.5 per cent. of the monoxide blood. Ten seedlings each of Lupinus albus were carefully measured and immersed in each of the above solutions and all of the plants were left in the dark at a temperature of 22° C. After eighteen hours it was found that the growth of the seedlings in normal blood gave an index of 75 per cent. Growth in 1 per cent. carbon monoxide blood gave an index of 37 per cent. Growth in 0.5 per cent. of carbon monoxide blood gave an index of 48 per cent. EXPERIMENT OCTOBER 9, 1925 Specimen of blood was obtained from a normal rabbit and a 1 per cent. solution was made. The rabbit was then allowed to inhale pure carbon monoxide until first signs of intoxication appeared. Blood was then drawn and it was found to contain about 30 per cent. of carbon monoxide hemoglobin. The growth of seedlings in a normal blood solution, and the blood obtained after inhaling carbon monoxide gave the following figures: Growth in normal blood 1 per cent. gave index of 72 per cent. Growth in carbon monoxide blood 1 per cent. gave index of 60 per cent. EXPERIMENT MARCH 31, 1927 A rat was killed with carbon monoxide gas. The relative indices of growth in 1 per cent. of normal rat blood and 1 per cent. of blood from the poisoned animal were as follows: Normal 75 per cent., carbon monoxide blood 47 per cent. EXPERIMENT APRIL 6, 1927 Pigeon was allowed to inhale carbon monoxide until convulsions occurred. Growth in solution of 1 per cent. of its blood was compared with growth in a 1 per cent. solution of normal pigeon's blood. The following results were obtained: Growth in normal blood 70 per cent., growth in carbon monoxide blood 50 per cent. Similar experiments were performed with bloods of men and several other animals. In each case it was found that the growth of Lupinus albus seedlings was markedly inhibited by solutions of carbon monoxide blood and also solutions of pure carbon monoxide hemoglobin. It was found that in cases of very sensitive preparations a definite inhibition in growth of seedlings could be noted in solutions of monoxide blood diluted to 0.01 per cent. when the preparations were kept in the dark and at a temperature of 20° C. The above observations speak in favor of poisonous effects produced by carbon monoxide or rather carbon monoxide hemoglobin apart from an interference with oxygenation processes. DAVID I. MACHT PHARMACOLOGICAL RESEARCH LABORATORY, HYNSON, WESTCOTT & DUNNING, ON THE THICKNESS OF THE HELMHOLTZ DOUBLE LAYER HELMHOLTZ and Lamb assumed the double electric layer around colloid particles to be of molecular dimensions, but Gouy calculated it to be greater and to increase with dilution of the solution. Whereas the diameter of a water molecule is about 0.03 x 10-6 cm, Gouy calculated the distance (a) between the surface of a colloid particle (having 10 electrostatic units of charge per sq. cm of surface) and the center of gravity of the excess ions of opposite sign in a 0.1 N solution of monovalent salt at 18° to be 0.096× 10-6 cm, and in a 0.001 N solution 0.96 × 10-6 cm. Gouy assumed a dielectric constant of 80. Burton 1 showed that Gouy's a was equal to Debye's: Bur K ton and Currie showed experimentally that the Helmholtz double layer was thick enough to account for repulsion between colloid particles as well as larger bodies (shot) and that its thickness increases with dilution. They account for the discharge of colloid particles on the addition of salts by the thinning of the double layer to the point of break-down of the dielectric. Frequency The capacity is most easily measured using alternating current, but since it changes slightly with frequency the electrostatic value can only be approximated. As will be seen from the table the thickness of the Helmholtz layer as measured is for 0.1 N solution 0.194 × 10-6 cm whereas Gouy calculated 0.096 × 10-6 cm, a fairly close agreement particularly in view of the uncertainty of the dielectric constant. The measured value for 0.001 N solution, 0.325 × 10-8 cm, is far from Gouy's value of 0.96 × 10-6 cm, but the change is in the right direction, i.e., increase in thickness of the Helmholtz layer with dilution of the electrolyte. Unit 10-6 cm T T in cycles per second 2 In previous papers (McClendon-1926) it has been shown that the double layer acts as a condenser and becomes thicker the greater the dilution of the solution. It was hoped that a method might be found to measure its thickness. Since the surface of a charged metal electrode (immersed in an electrolyte solution) and the layer of excess ions of opposite sign act as the plates of a condenser whose capacity may be measured, knowing the dielectric constant, the thickness of the Helmholtz double layer may be calculated. The capacity KS in microfarads, C=0.0885 × 10-6 where S is the T surface area of the electrode in cm2, T is the thickness of the dielectric (Helmholtz double layer) and |